Methods for Preserving and Administering Pre-Beta High Density Lipoprotein Extracted from Human Plasma

ABSTRACT

Systems and methods for acquiring, preserving, and administering delipidated plasma. Extracted delipidated plasma, comprising pre-beta HDL, is obtained and are spot tested to establish baseline amounts or concentrations of pre-beta HDL. The batches are subjected to preservation, stored, and then prepared again for use at some later date. A portion of the batch may be tested again to determine if the pre-beta HDL in the delipidated plasma has degraded or is no longer effective.

CROSS-REFERENCE

The present application relies on U.S. Provisional Patent ApplicationNo. 62/611,098, entitled “Methods for Treating Cholesterol-RelatedDiseases” and filed on Dec. 28, 2017, which is herein incorporated byreference in its entirety.

FIELD

The present invention generally relates to systems, apparatus andmethods for removing lipids from HDL particles while leaving LDLparticles substantially intact, via the extracorporeal treatment ofblood plasma using either a single solvent or multiple solvents, inorder to treat chronic cardiovascular diseases and acute renal diseases.More specifically, the present invention relates to systems and methodsfor preserving and administering pre-β HDL from non-autologous,delipidated plasma.

BACKGROUND

Familial Hypercholesterolemia (FH) is an inherited genetic autosomaldominant disease characterized by markedly elevated low densitylipoprotein (LDL), tendon xanthomas, and premature coronary heartdisease, caused by mutations of “FH genes,” which include theLDL-receptor (LDLR), apolipoprotein B-100 (ApoB) or proproteinconvertase subtilisin/kexin type 9 (PCSK9). FH produces a clinicallyrecognizable pattern that consists of severe hypercholesterolemia due tothe accumulation of LDL in the plasma, cholesterol deposition in tendonsand skin, as well as a high risk of atherosclerosis manifesting almostexclusively as coronary artery disease (CAD). In FH patients, thisgenetic mutation makes the liver unable to effectively metabolize (orremove) excess plasma LDL, resulting in increased LDL levels.

If an individual has inherited a defective FH gene from one parent, theform of FH is called Heterozygous FH. Heterozygous FH is a commongenetic disorder, inherited in an autosomal dominant pattern, occurringin approximately 1:500 people in most countries. If the individual hasinherited a defective FH gene from both parents, the form of FH iscalled Homozygous FH. Homozygous FH is very rare, occurring in about 1in 160,000 to one million people worldwide, and results in LDLlevels >700 mg/dl, 10 fold higher than the ideal 70 mg/dl level desiredfor patients with CVD. Due to the high LDL levels, patients withHomozygous FH have aggressive atherosclerosis (narrowing and blocking ofblood vessels) and early heart attacks. This process starts before birthand progresses rapidly. It can affect the coronary arteries, carotidarteries, aorta, and aortic valve.

Heterozygous FH (HeFH) is normally treated with statins, bile acidsequestrants, or other lipid lowering agents that lower cholesterollevels, and/or by offering genetic counseling. Homozygous FH (HoFH)often does not respond adequately to medical therapy and may requireother treatments, including LDL apheresis (removal of LDL in a methodsimilar to dialysis), ileal bypass surgery to dramatically lower theirLDL levels, and occasionally liver transplantation. A few medicationshave recently been approved for use by HoFH subjects. However, thesemedications lower LDL only, and modestly contribute to slowing, but notstopping, further progression of atherosclerosis. Additionally, thesemedications are known to have significant side-effects.

Cholesterol is synthesized by the liver or obtained from dietarysources. LDL is responsible for transferring cholesterol from the liverto tissues at different sites in the body. However, if LDL collects onthe arterial walls, it undergoes oxidation caused by oxygen freeradicals liberated from the body's chemical processes and interactsdeleteriously with the blood vessels. The modified LDL causes whiteblood cells in the immune system to gather at the arterial walls,forming a fatty substance called plaque and injuring cellular layersthat line blood vessels. The modified oxidized LDL also reduces thelevel of nitric oxide, which is responsible for relaxing the bloodvessels and thereby allowing the blood to flow freely. As this processcontinues, the arterial walls slowly constrict, resulting in hardeningof the arteries and thereby reducing blood flow. The gradual build-up ofplaque can result in blockage of a coronary vessel and ultimately in aheart attack. The plaque build up can also occur in peripheral vesselssuch as the legs and this condition is known as peripheral arterialdisease.

Obstructions can also appear in blood vessels that supply blood to thebrain, which can result in ischemic strokes. The underlying conditionfor this type of obstruction is the development of fatty deposits liningthe vessel walls. It is known that at least 2.7% of men and women overthe age of 18 in the United States have a history of stroke. Prevalenceof stroke is also known to be higher with increasing age. With theincrease in the aging population, the prevalence of stroke survivors isprojected to increase, especially among elderly women. A considerableportion of all strokes (at least 87%) are ischemic in nature.

Further, it has been shown that hypercholesterolemia and inflammationare two dominant mechanisms implicated in the development ofatherosclerosis. There is significant overlap between vascular riskfactors for both Alzheimer's disease and atherosclerosis. Inflammationhas been implicated in Alzheimer's disease pathogenesis and it issuggested that abnormalities in cholesterol homeostasis may have a roleas well. In addition, many of the contributory factors in atherogenesisalso contribute to Alzheimer's disease. Specifically, in cell cultures,increased and decreased cholesterol levels promote and inhibit theformation of beta amyloid (Aβ) from Amyloid Precursor Protein (APP),respectively. Thus, the use of treatments with proven effects on theprocess of atherosclerosis may be one method for treating theprogression of the Alzheimer's disease.

Another common cardiovascular disease that occurs due to development ofatherosclerosis (hardening and narrowing of the arteries) within theelastic lining inside a coronary artery, is Coronary Artery Disease(CAD), also known as Ischemic Heart Disease (IHD). On the basis of astatistical data collected from 2009 to 2012, an estimated 15.5 millionAmericans≥20 years of age have CAD. The total CAD prevalence in theUnited States is 6.2% of adults ≥20 years of age.

An acute decrease in blood flow in the coronary arteries may result inpart of the heart muscle unable to function properly. This condition isknown as Acute Coronary Syndrome (ACS). A conservative estimate for thenumber of hospital discharges with ACS in 2010 is 625,000.

In contrast to LDL, high plasma HDL levels are desirable because theyplay a major role in “reverse cholesterol transport”, where the excesscholesterol is transferred from tissue sites to the liver where it iseliminated. Optimal total cholesterol levels are 200 mg/dl or below witha LDL cholesterol level of 160 mg/dl or below and a HDL-cholesterollevel of 45 mg/dl for men and 50 mg/dl for women. Lower LDL levels arerecommended for individuals with a history of elevated cholesterol,atherosclerosis or coronary artery disease. High levels of LDL increasethe lipid content in coronary arteries resulting in formation of lipidfilled plaques that are vulnerable to rupture. On the other hand, HDLhas been shown to decrease the lipid content in the lipid filledplaques, reducing the probability of rupture. In the last several years,clinical trials of low density lipoprotein (LDL)-lowering drugs havedefinitively established that reductions in LDL are associated with a30-45% decrease in clinical cardiovascular disease (CVD) events. CVDevents include events occurring in diseases such as HoFH, HeFH, andperipheral arterial disease. Despite lowered LDL, however, many patientscontinue to have cardiac events. Low levels of HDL are often present inhigh risk subjects with CVD, and epidemiological studies have identifiedHDL as an independent risk factor that modulates CVD risk. In additionto epidemiologic studies, other evidence suggests that raising HDL wouldreduce the risk of CVD. There has been increasing interest in changingplasma HDL levels by dietary, pharmacological or genetic manipulationsas a potential strategy for the treatment of CVD including HoFH, HeFH,Ischemic stroke, CAD, ACS, and peripheral arterial disease and fortreating the progression of Alzheimer's Disease.

The protein component of LDL, known as apolipoprotein-B (ApoB), and itsproducts, comprise atherogenic elements. Elevated plasma LDL levels andreduced HDL levels are recognized as primary causes of coronary disease.ApoB is in highest concentration in LDL particles and is not present inHDL particles. Apolipoprotein A-I (ApoA-I) and apolipoprotein A-II(ApoA-II) are found in HDL. Other apolipoproteins, such as ApoC and itssubtypes (C-I, C-II and C-III), ApoD, and ApoE are also found in HDL.ApoC and ApoE are also observed in LDL particles.

Numerous major classes of HDL particles including HDL2b, HDL2a, HDL3a,HDL3b and HDL3 have been reported. Various forms of HDL particles havebeen described on the basis of electrophoretic mobility on agarose astwo major populations, a major fraction with α-HDL mobility and a minorfraction with migration similar to VLDL. This latter fraction has beencalled pre-β HDL and these particles are the most efficient HDL particlesubclass for inducing cellular cholesterol efflux.

The HDL lipoprotein particles are comprised of ApoA-I, phospholipids andcholesterol. The pre-β HDL particles are considered to be the firstacceptors of cellular free cholesterol and are essential in eventuallytransferring free and esterified cholesterol to α-HDL. Pre-β HDLparticles may transfer cholesterol to α-HDL or be converted to α-HDL.The alpha HDL transfers cholesterol to the liver, where excesscholesterol can be removed from the body.

HDL levels are inversely correlated with atherosclerosis and coronaryartery disease. Once cholesterol-carrying α-HDL reaches the liver, theα-HDL particles divest of the cholesterol and transfer the freecholesterol to the liver. The α-HDL particles (divested of cholesterol)are subsequently converted to pre-β HDL particles and exit the liver,which then serve to pick up additional cholesterol within the body andare converted back to α-HDL, thus repeating the cycle. Accordingly, whatis needed is a method to decrease or remove cholesterol from thesevarious HDL particles, especially the α-HDL particles, so that they areavailable to remove additional cholesterol from cells.

Renal arterial stenosis refers to a blockage in an artery that suppliesblood to the kidney and is characterized in two forms: a) smooth muscleplaque or b) cholesterol filled plaque. This condition, generally knownas renal arterial stenosis, decreases blood flow to the kidney and canresult in high blood pressure. Plaque in the renal arteries may bediscovered during a CT angiogram. In some cases, renal arterial stenosisis discovered while performing a CT angiogram for an aortic aneurysm.Conventionally, blood pressure increases gradually with age. However, asudden onset of hypertension is also likely to be associated with renalobstruction or renal arterial stenosis. A decrease in flow of blood tothe kidney causes vasoconstriction or high blood pressure, as the kidneystarts producing an excess of cytokines.

In addition, a “cholesterol embolism’, can occur when the cholesterol inthe artery is released, usually from an atherosclerotic plaque, andtravels as an embolus in the bloodstream causing an obstruction (as anembolism) in blood vessels that are positioned further away. Once incirculation, the cholesterol particles get stuck in tiny blood vessels,or arterioles. They can reduce blood flow to tissues and causeinflammation and tissue damage that can harm the kidneys. A cholesterolembolism may result in renal failure, and is a disease state referred toas Atheroembolic Renal Disease (AERD). AERD is one of the manifestationsof diseases that may occur due to a cholesterol-filled plaque. In apatient with AERD, the plaque may rupture in the artery and release thecholesterol and other “junk” within the plaque into the vessel. Thereleased cholesterol and junk may travel down the artery and may blockthe artery and injure a part of the kidney and its tissues, therebyresulting in AERD. Atherosclerosis of the aorta is the most common causeof AERD.

Currently, treatment of renal arterial stenosis, its manifestations suchas AERD, and other cardiovascular diseases involves putting a stent inan artery to open the vessel. This technique often normalizes the bloodpressure. However, installing a stent is likely to only treat thesymptoms, such as high blood pressure. There are also instances when theblood pressure is normal, but AERD is present in a patient. There isthus a need to address the underlying cause of the disease, and treatrenal arterial stenosis either in combination with or independently ofhigh blood pressure symptoms.

Hyperlipidemia (or abnormally high concentration of lipids in the blood)may be treated by changing a patient's diet. However, diet as a primarymode of therapy requires a major effort on the part of patients,physicians, nutritionists, dietitians, and other health careprofessionals and thus undesirably taxes the resources of healthprofessionals. Another negative aspect of this therapy is that itssuccess does not rest exclusively on diet. Rather, success of dietarytherapy depends upon a combination of social, psychological, economic,and behavioral factors. Thus, therapy based only on correcting flawswithin a patient's diet, is not always successful.

In instances when dietary modification has been unsuccessful, drugtherapy has been used as adjunctive therapy. Such therapy has includeduse of commercially available hypolipidemic drugs administered alone orin combination with other therapies as a supplement to dietary control.These drugs, called statins, include lovastatin, pravastatin,simvastatin, fluvastatin, atorvastatin, and cerivastatin. Statins areparticularly effective for lowering LDL levels and are also effective inthe reduction of triglycerides, apparently in direct proportion to theirLDL-lowering effects. Statins raise HDL levels, but to a lesser extentthan other anti-cholesterol drugs. Statins also increase nitric oxide,which, as described above, is reduced in the presence of oxidized LDL.

Bile acid resins, another drug therapy, work by binding with bile acid,a substance made by the liver using cholesterol as one of the primarymanufacturing components. Because the drugs bind with bile acids in thedigestive tract, they are then excreted with the feces rather than beingabsorbed into the body. The liver, as a result, must take morecholesterol from the circulation to continue constructing bile acids,resulting in an overall decrease in LDL levels.

Nicotinic acid, or niacin, also known as vitamin B3, is effective inreducing triglyceride levels and raising HDL levels higher than anyother anti-cholesterol drug. Nicotinic acid also lowers LDL-cholesterol.

Fibric acid derivatives, or fibrates, are used to lower triglyceridelevels and increase HDL when other drugs ordinarily used for thesepurposes, such as niacin, are not effective.

Probucol lowers LDL-cholesterol levels, however, it also lowers HDLlevels. It is generally used for certain genetic disorders that causehigh cholesterol levels, or in cases where other cholesterol-loweringdrugs are ineffective or cannot be used.

PCSK9s lower LDL-cholesterol levels via increasing the cellular level ofLDL receptors that reside in the liver.

Hypolipidemic drugs have had varying degrees of success in reducingblood lipid; however, none of the hypolipidemic drugs successfullytreats all types of hyperlipidemia. While some hypolipidemic drugs havebeen fairly successful, the medical community has found littleconclusive evidence that hypolipidemic drugs cause regression ofatherosclerosis. In addition, all hypolipidemic drugs have undesirableside effects. As a result of the lack of success of dietary control,drug therapy and other therapies, atherosclerosis remains a major causeof death in many parts of the world.

New therapies have been used to reduce the amount of lipid in patientsfor whom drug and diet therapies were not sufficiently effective. Forexample, extracorporeal procedures like plasmapheresis and LDL-apheresishave been employed and are shown to be effective in lowering LDL.

Plasmapheresis therapy or plasma exchange therapy, involves replacing apatient's plasma with donor plasma or more usually a plasma proteinfraction. Plasmapheresis is a process whereby the blood plasma isremoved from blood cells by a cell separator. The separator works eitherby spinning the blood at high speed to separate the cells from the fluidor by passing the blood through a membrane with pores so small that onlythe fluid component of the blood can pass through. The cells arereturned to the person undergoing treatment, while the plasma isdiscarded and replaced with other fluids.

This treatment has resulted in complications due to the introduction offoreign proteins and transmission of infectious diseases. Further,plasmapheresis has the disadvantage of non-selective removal of allserum lipoproteins, such as VLDL, LDL, and HDL. Moreover, plasmapheresiscan result in several side effects including allergic reactions in theform of fever, chills, and rash and possibly even anaphylaxis.

As described above, it is not desirable to remove HDL, which is secretedfrom both the liver and the intestine as nascent, disk-shaped particlesthat contain cholesterol and phospholipids. HDL is believed to play arole in reverse cholesterol transport, which is the process by whichexcess cholesterol is removed from tissues and transported to the liverfor reuse or disposal in the bile.

In contrast to plasmapheresis, the LDL-apheresis procedure selectivelyremoves ApoB containing cholesterol, such as LDL, while retaining HDL.Several methods for LDL-apheresis have been developed. These techniquesinclude absorption of LDL in heparin-agarose beads, the use ofimmobilized LDL-antibodies, cascade filtration absorption to immobilizedextran sulfate, and LDL precipitation at low pH in the presence ofheparin. Each method described above is effective in removing LDL. Thistreatment process has disadvantages, however, including the failure topositively affect HDL or to cause a metabolic shift that can enhanceatherosclerosis and other cardiovascular diseases. LDL apheresis, as itsname suggests, merely treats LDL in patients with severe hyperlipidemia.

Yet another method of achieving a reduction in plasma cholesterol inhomozygous familial hypercholesterolemia, heterozygous familialhypercholesterolemia and patients with acquired hyperlipidemia is anextracorporeal lipid elimination process, referred to as cholesterolapheresis. In cholesterol apheresis, blood is withdrawn from a patient,the plasma is separated from the blood, and the plasma is mixed with asolvent mixture. The solvent mixture extracts lipids from the plasma.Thereafter, the delipidated plasma is recombined with the patient'sblood cells and returned to the patient. Using this procedure, however,results in a modification of the LDL particles, such that the modifiedLDL particles could result in increased intensity of the heart disease.At the same time, this process also resulted in further delipidation ofthe HDL particles.

Conventional extracorporeal delipidation processes, however, aredirected toward the concurrent delipidation of LDL and HDL. This processcan have a number of disadvantages. The main disadvantage being thatdelipidated LDL tends to aggregate and subsequently cause an increase inheart disease conditions, rather than decrease. In addition,extracorporeal systems are designed to subject body fluid volumes tosubstantial processing, possibly through multiple stage solvent exposureand extraction steps.

Vigorous multi-stage solvent exposure and extraction can have severaldrawbacks. It may be difficult to remove a sufficient amount of solventsfrom the delipidated plasma in order for the delipidated plasma to besafely returned to a patient.

Hence, existing apheresis and extracorporeal systems for treatment ofplasma constituents suffer from a number of disadvantages that limittheir ability to be used in clinical applications. A need exists forimproved systems, apparatuses and methods capable of removing lipidsfrom blood components in order to provide treatments and preventativemeasures for chronic cardiovascular diseases. Methods have also beenprovided to selectively remove lipid from HDL particles and therebycreate modified HDL particles with increased capacity to acceptcholesterol.

Methods have been provided to selectively remove lipid from HDLparticles and thereby create modified HDL particles with increasedcapacity to accept cholesterol, without substantially affecting LDLparticles, in chronic diseases. However, these methods envision theimmediate re-administration of the modified HDL particles and do notprovide any means to preserve, store, or otherwise use the modified HDLparticles over longer periods of time.

What is also needed is a method to preserve delipidated plasma and themodified HDL particles. Plasma derived from autologous andnon-autologous sources are required to be introduced to a patient withina few hours of the derivation. However, there are situations when it maynot be possible to introduce derived modified HDL particles within thestipulated time period of the derivation to a patient. This mayparticularly be true in cases where a patient needs modified HDLparticles that cannot be sufficiently derived from the patient(autologous), but rather, non-autologous plasma sources are available.Access to life-saving therapy for more patients may be enhanced bypreserving and making delipidated plasma readily available, so that itcan be used as and when required.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods, which aremeant to be exemplary and illustrative, not limiting in scope.

A method for preserving pre-beta high density lipoprotein foradministration to a patient, comprising: obtaining a batch ofdelipidated plasma comprising the pre-beta high density lipoprotein;testing a portion of the batch of the delipidated plasma to characterizethe pre-beta high density lipoprotein; preserving the batch of thedelipidated plasma; preparing the preserved delipidated plasma foradministration to the patient; testing the prepared delipidated plasmato characterize the pre-beta high density lipoprotein; and administeringthe pre-beta high density lipoprotein to the patient.

Optionally, the method further comprises, prior to preserving, modifyingan amount of the pre-beta high density lipoprotein to insure aconcentration of the pre-beta high density lipoprotein is in a range of1 mg/dl to 400 mg/dl.

Optionally, preserving comprises freezing the batch at a temperatureless than −30° C.

Optionally, preparing comprises thawing the preserved delipidated plasmain a temperature range of 2° C. to 26° C.

Optionally, preserving comprises subjecting a volume of delipidatedplasma in a range from 1 milliliter to 2 liters to a temperature lessthan −30° C. for less than 20 minutes.

Optionally, testing the portion of the batch of the delipidated plasmato characterize the pre-beta high density lipoprotein comprisesdetermining a first concentration of the pre-beta high densitylipoprotein. Optionally, testing the prepared delipidated plasma tocharacterize the pre-beta high density lipoprotein comprises determininga second concentration of the pre-beta high density lipoprotein andcomparing the second concentration of the pre-beta high densitylipoprotein to the first concentration of the pre-beta high densitylipoprotein to determine an extent of degradation. Optionally, themethod further comprises determining if the prepared delipidated plasmais suitable for administration based on the second concentration of thepre-beta high density lipoprotein.

Optionally, the method further comprises, prior to preservation, addinga preservative to the delipidated plasma.

Optionally, preparing comprises thawing the preserved delipidated plasmaand further comprising storing the thawed delipidated plasma at atemperature in a range of 1° C. to 6° C. for no more than 5 days.

The present specification also discloses a method for preservingmodified high density lipoproteins for administration to a patient,comprising: obtaining a batch of delipidated plasma comprising themodified high density lipoproteins by connecting at least one person toa device for withdrawing blood, withdrawing blood containing blood cellsfrom the at least one person, separating the blood cells from the bloodto yield a blood plasma fraction containing high density lipoproteinsand low density lipoproteins, delipidating the high density lipoproteinsusing a solvent, separating out the low density lipoproteins, andcollecting the delipidated plasma with the modified high densitylipoproteins; testing a portion of the batch of the delipidated plasmato characterize the modified high density lipoproteins; preserving thebatch of the delipidated plasma; preparing the preserved delipidatedplasma for administration to the patient; testing the prepareddelipidated plasma to characterize the modified high densitylipoproteins; and administering the modified high density lipoproteinsto the patient.

Optionally, the method further comprises, prior to preserving, modifyingan amount of the modified high density lipoproteins to insure aconcentration of the modified high density lipoproteins is in a range of1 mg/dl to 400 mg/dl.

Optionally, preserving comprises freezing the batch at a temperatureless than −30° C.

Optionally, preparing comprises thawing the preserved delipidated plasmain a temperature range of 2° C. to 26° C.

Optionally, preserving comprises subjecting a volume of delipidatedplasma in a range from 1 milliliter to 2 liters to a temperature lessthan −30° C. for less than 20 minutes.

Optionally, testing the portion of the batch of the delipidated plasmato characterize the modified high density lipoproteins comprisesdetermining a first concentration of the modified high densitylipoproteins. Optionally, testing the prepared delipidated plasma tocharacterize the modified high density lipoproteins comprisesdetermining a second concentration of the modified density lipoproteinsand comparing the second concentration of the modified high densitylipoproteins to the first concentration of the modified densitylipoproteins to determine an extent of degradation. Optionally, themethod further comprises determining if the prepared delipidated plasmais suitable for administration based on the second concentration of themodified high density lipoproteins.

Optionally, the method further comprises, prior to preservation, addinga preservative to the delipidated plasma.

Optionally, preparing comprises thawing the preserved delipidated plasmaand further comprising storing the thawed delipidated plasma at atemperature in a range of 1° C. to 6° C. for no more than 5 days.

The present specification also discloses a method for treatment ofcardiovascular disease in a patient, comprising: obtaining a bloodplasma fraction containing high density lipoprotein and low densitylipoprotein; mixing the plasma fraction with a lipid removing agentwhich removes lipids to yield a mixture of lipid, the lipid removingagent, modified high density lipoprotein, and the low densitylipoprotein, wherein the modified high density lipoprotein is adelipidated high density lipoprotein; separating the modified highdensity lipoprotein and the low density lipoprotein from the lipid andthe lipid removing agent; preserving the modified high densitylipoprotein for a prolonged period of time; preparing for use thepreserved modified high density lipoprotein; separating components ofhigh density lipoprotein particles from the modified high densitylipoprotein prepared after preservation; and delivering the componentsof high density lipoprotein particles to the patient.

Optionally, the method of preserving comprises freezing. Optionally, themethod of preparing for use comprises thawing.

Optionally, the method of preserving comprises preserving a volume of DPranging from 1 milliliter to 2 liters.

Optionally, the method of mixing comprises mixing the blood plasmafraction with a lipid removing agent which removes lipids associatedwith the high density lipoprotein without substantially modifying thelow density lipoprotein.

Optionally, the method of obtaining a blood plasma fraction containinghigh density lipoprotein and low density lipoprotein comprises obtainingfrom at least one of the patient or an individual other than thepatient.

Optionally, the method for treatment of cardiovascular disease includestreating at least one of AERD, Homozygous Familial HypercholesterolemiaHeterozygous Familial Hypercholesterolemia, Ischemic stroke, CoronaryArtery Disease, Acute Coronary Syndrome, and peripheral arterialdisease.

Optionally, the method for treatment of cardiovascular diseases includesmethod for treatment of a progression of Alzheimer's disease.

Optionally, the step of mixing the blood plasma fraction with a lipidremoving agent yields modified high density lipoprotein that has anincreased concentration of pre-beta high density lipoprotein relative tototal protein.

Optionally, the step of obtaining a blood plasma fraction from treatingcardiovascular diseases further comprises: connecting a person to adevice for withdrawing blood; withdrawing blood containing blood cellsfrom the person; and separating the blood cells from the blood to yielda blood plasma fraction containing high density lipoprotein and lowdensity lipoprotein.

Optionally, the step of separating components of high densitylipoprotein particles from the modified high density lipoproteincomprises using affinity chromatography.

Optionally, the method of using affinity chromatography comprises:adding the plasma containing delipidated high density lipoprotein to acolumn; allowing the plasma to drip through the column wherein thecolumn contains an antibody for binding to ApoA-I protein; washing thecolumn to remove any unwanted material; and delivering a disassociatingreagent through the column to break a bond between the antibody andApoA-I protein, thereby separating at least pre-beta HDL.

Optionally, the step of separating components of high densitylipoprotein particles from the modified high density lipoproteincomprises using ultracentrifugation.

Optionally, using ultracentrifugation comprises: spinning the modifiedhigh density lipoprotein at a density of 1.21; separating out a bottomfraction containing pre-beta high density lipoprotein particles andplasma proteins; and spinning the bottom fraction at a density of 1.25to separate pre-beta high density lipoprotein particles from plasmaproteins.

Optionally, using ultracentrifugation comprises: spinning the modifiedhigh density lipoprotein at a density of 1.006; separating bottomfraction containing plasma with low density lipoprotein and high densitylipoprotein; spinning the separated plasma with low density lipoproteinand high density lipoprotein at a density of 1.063; separating out abottom fraction containing high density lipoprotein particles; spinningthe separated high density lipoprotein particles at a density of 1.21;separating out a bottom fraction containing pre-beta high densitylipoprotein particles and plasma proteins; and spinning the bottomfraction at a density of 1.25 to separate pre-beta high densitylipoprotein particles from plasma proteins.

Optionally, using ultracentrifugation comprises: spinning the modifiedhigh density lipoprotein at a density of 1.063; separating out a bottomfraction containing alpha and pre-beta high density lipoproteinparticles and plasma proteins; and spinning the bottom fraction at adensity of 1.25 to separate alpha and pre-beta high density lipoproteinparticles from plasma proteins.

The present specification also discloses a method for treatment ofcardiovascular disease in a patient, comprising: obtaining a bloodplasma fraction containing high density lipoprotein and low densitylipoprotein; mixing the plasma fraction with a lipid removing agentwhich removes lipids to yield a mixture of lipid, the lipid removingagent, modified high density lipoprotein, and the low densitylipoprotein, wherein the modified high density lipoprotein is adelipidated high density lipoprotein; separating the modified highdensity lipoprotein and the low density lipoprotein from the lipid andthe lipid removing agent; separating components of high densitylipoprotein particles from the modified high density lipoprotein;preserving the components of high density lipoprotein particles for aprolonged period of time; preparing for use the preserved components ofhigh density lipoprotein particles; and delivering the components ofhigh density lipoprotein particles to the patient.

The present specification also discloses a method for treatment ofcardiovascular disease in a patient, comprising: obtaining a bloodplasma fraction containing high density lipoprotein and low densitylipoprotein; mixing the plasma fraction with a lipid removing agentwhich removes lipids to yield a mixture of lipid, the lipid removingagent, modified high density lipoprotein, and the low densitylipoprotein, wherein the modified high density lipoprotein is adelipidated high density lipoprotein; separating the modified highdensity lipoprotein and the low density lipoprotein from the lipid andthe lipid removing agent; separating components of high densitylipoprotein particles from the modified high density lipoprotein; andpreserving the components of high density lipoprotein particles for aprolonged period of time.

Optionally, the method further comprises preparing for use the preservedcomponents of high density lipoprotein particles.

Optionally, the method further comprises delivering the components ofhigh density lipoprotein particles to the patient.

Optionally, the step of preservation comprises freezing.

Optionally, the step of preparing comprises thawing.

Optionally, the components of high density lipoprotein particlescomprise alpha high density lipoprotein and/or pre-beta high densitylipoprotein.

Optionally, the components of high density lipoprotein particlescomprise pre-beta high density lipoprotein.

The present specification also discloses a method for treatment ofcardiovascular disease in a patient, comprising: obtaining a bloodplasma fraction containing high density lipoprotein and low densitylipoprotein; mixing the plasma fraction with a lipid removing agentwhich removes lipids to yield a mixture of lipid, the lipid removingagent, modified high density lipoprotein, and the low densitylipoprotein, wherein the modified high density lipoprotein is adelipidated high density lipoprotein; separating the modified highdensity lipoprotein and the low density lipoprotein from the lipid andthe lipid removing agent; preserving the modified high densitylipoprotein for a predetermined or prolonged period of time; preparingfor use the preserved modified high density lipoprotein; separatingcomponents of high density lipoprotein particles from the modified highdensity lipoprotein prepared after preservation; preserving thecomponents of high density lipoprotein particles for a prolonged orpredetermined period of time; preparing for use the preserved componentsof high density lipoprotein particles; and delivering the components ofhigh density lipoprotein particles to the patient.

Optionally, the preserving comprises freezing.

Optionally, the preparing comprises thawing.

Optionally, the components of high density lipoprotein particlescomprise alpha high density lipoprotein and/or pre-beta high densitylipoprotein.

Optionally, the components of high density lipoprotein particlescomprise pre-beta high density lipoprotein.

The aforementioned and other embodiments of the present specificationshall be described in greater depth in the drawings and detaileddescription provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will beappreciated, as they become better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings, wherein:

FIG. 1A is a flow chart illustrating an exemplary process for separatingpre-beta HDL from delipidated (modified) HDL, in accordance with someembodiments of the present specification;

FIG. 1B is a schematic representation of a system comprising a pluralityof components used in accordance with some embodiments of the presentspecification to achieve the processes disclosed herein;

FIG. 1C is a pictorial illustration of an exemplary embodiment of asystem configuration of a plurality of components used in accordancewith some embodiments of the present specification to achieve theprocesses disclosed herein;

FIG. 1D is a flow chart illustrating an exemplary process used forincreasing the concentration of desired substances in delipidated plasmausing affinity chromatography, in accordance with some embodiments ofthe present specification;

FIG. 1E is a flow chart illustrating an exemplary process used forincreasing the concentration of desired substances in delipidatedplasma, using ultracentrifugation, in accordance with some embodimentsof the present specification;

FIG. 1F is a flow chart illustrating another exemplary process used forincreasing the concentration of desired substances in delipidatedplasma, using ultracentrifugation, in accordance with some embodimentsof the present specification;

FIG. 1G is a flow chart illustrating another exemplary set of steps thatare used to increase the concentration of desired substances in thetreated plasma, using ultracentrifugation, in accordance with someembodiments of the present specification; and

FIG. 2 is a flow chart illustrating a process for testing, preserving,and validating stored delipidated plasma comprising pre-beta HDL.

DETAILED DESCRIPTION

In some embodiments, the present specification is directed towardssystems, apparatuses and methods for preserving modified HDL particles(also referred to as delipidated HDL) with reduced lipid content,particularly reduced cholesterol content derived primarily from plasmaof non-autologous sources for a patient, where the preserved product maybe for later use. Embodiments of the present specification create andpreserve these modified HDL particles with reduced lipid content withoutsubstantially modifying LDL particles. Embodiments of the presentspecification modify original α-HDL particles (present in delipidatedplasma) to yield modified HDL particles that have an increasedconcentration of components of HDL, including α-HDL and/or pre-β HDLrelative to the original HDL. Further, the newly formed derivatives ofHDL particles (modified HDL) are treated to separate α-HDL and/or pre-βHDL, from the delipidated plasma. In some embodiments, delipidatedplasma is treated to create an even more concentrated solution of α-HDLand/or pre-β HDL. The modified HDL, with a concentrated solution ofα-HDL and/or pre-β HDL is preserved, in an embodiment, by freezing andadministered to the patient at a later time after thawing the preservedmodified HDL, in order to enhance cellular cholesterol efflux and treatcardiovascular diseases and/or other lipid-associated diseases. In anembodiment, the modified HDL contains a concentrated solution ofapproximately 20% α-HDL particles (present in delipidated plasma) andapproximately 80% pre-β HDL.

The treatment processes of the present specification renders the methodsand systems of the present specification more effective in treatingcardiovascular diseases including Homozygous FamilialHypercholesterolemia (HoFH), Heterozygous Familial Hypercholesterolemia(HeFH), Ischemic stroke, Coronary Artery Disease (CAD), Acute CoronarySyndrome (ACS), peripheral arterial disease (PAD), AERD, Renal ArterialStenosis (RAS) and its manifestations, and for treating the progressionof Alzheimer's Disease.

The present specification is directed towards multiple embodiments. Thefollowing disclosure is provided in order to enable a person havingordinary skill in the art to practice the invention. Language used inthis specification should not be interpreted as a general disavowal ofany one specific embodiment or used to limit the claims beyond themeaning of the terms used therein. The general principles defined hereinmay be applied to other embodiments and applications without departingfrom the spirit and scope of the invention. Also, the terminology andphraseology used is for the purpose of describing exemplary embodimentsand should not be considered limiting. Thus, the present invention is tobe accorded the widest scope encompassing numerous alternatives,modifications and equivalents consistent with the principles andfeatures disclosed. For purpose of clarity, details relating totechnical material that is known in the technical fields related to theinvention have not been described in detail so as not to unnecessarilyobscure the present invention. In the description and claims of theapplication, each of the words “comprise” “include” and “have”, andforms thereof, are not necessarily limited to members in a list withwhich the words may be associated.

It should be noted herein that any feature or component described inassociation with a specific embodiment may be used and implemented withany other embodiment unless clearly indicated otherwise.

The term “fluid” may be defined as fluids from animals or humans thatcontain lipids or lipid containing particles, fluids from culturingtissues and cells that contain lipids and fluids mixed withlipid-containing cells. For purposes of this invention, decreasing theamount of lipids in fluids includes decreasing lipids in plasma andparticles contained in plasma, including but not limited to HDLparticles. Fluids include, but are not limited to: biological fluids;such as blood, plasma, serum, lymphatic fluid, cerebrospinal fluid,peritoneal fluid, pleural fluid, pericardial fluid, various fluids ofthe reproductive system including, but not limited to, semen,ejaculatory fluids, follicular fluid and amniotic fluid; cell culturereagents such as normal sera, fetal calf serum or serum derived from anyanimal or human; and immunological reagents, such as variouspreparations of antibodies and cytokines from culturing tissues andcells, fluids mixed with lipid-containing cells, and fluids containinglipid-containing organisms, such as a saline solution containinglipid-containing organisms. A preferred fluid treated with the methodsof the present invention is plasma.

The term “lipid” may be defined as any one or more of a group of fats orfat-like substances occurring in humans or animals. The fats or fat-likesubstances are characterized by their insolubility in water andsolubility in organic solvents. The term “lipid” is known to those ofordinary skill in the art and includes, but is not limited to, complexlipid, simple lipid, triglycerides, fatty acids, glycerophospholipids(phospholipids), true fats such as esters of fatty acids, glycerol,cerebrosides, waxes, and sterols such as cholesterol and ergosterol.

The term “extraction solvent” may be defined as one or more solventsused for extracting lipids from a fluid or from particles within thefluid. This solvent enters the fluid and remains in the fluid untilremoved by other subsystems. Suitable extraction solvents includesolvents that extract or dissolve lipid, including but not limited tophenols, hydrocarbons, amines, ethers, esters, alcohols,halohydrocarbons, halocarbons, and combinations thereof. Examples ofsuitable extraction solvents are ethers, esters, alcohols,halohydrocarbons, or halocarbons which include, but are not limited todi-isopropyl ether (DIPE), which is also referred to as isopropyl ether,diethyl ether (DEE), which is also referred to as ethyl ether, lowerorder alcohols such as butanol, especially n-butanol, ethyl acetate,dichloromethane, chloroform, isoflurane, sevoflurane(1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy) propane-d3),perfluorocyclohexanes, trifluoroethane, cyclofluorohexanol, andcombinations thereof.

The term “patient” refers to animals and humans, which may be either afluid source to be treated with the methods of the present invention ora recipient of derivatives of HDL particles and or plasma with reducedlipid content.

The term “HDL particles” encompasses several types of particles definedbased on a variety of methods such as those that measure charge,density, size and immuno-affinity, including but not limited toelectrophoretic mobility, ultracentrifugation, immunoreactivity andother methods known to one of ordinary skill in the art. Such HDLparticles include but are not limited to the following: α-HDL, pre-β HDL(including pre-β1 HDL, pre-β2 HDL and pre-β3HDL), HDL2 (including HDL2aand HDL2b), HDL3, VHDL, LpA-I, LpA-II, LpA-I/LpA-II (for a review seeBarrans et al., Biochemica Biophysica Acta 1300; 73-85,1996).Accordingly, practice of the methods of the present invention createsmodified HDL particles. These modified derivatives of HDL particles maybe modified in numerous ways including but not limited to changes in oneor more of the following metabolic and/or physico-chemical properties(for a review see Barrans et al., Biochemica Biophysica Acta 1300;73-85,1996); molecular mass (kDa); charge; diameter; shape; density;hydration density; flotation characteristics; content of cholesterol;content of free cholesterol; content of esterified cholesterol; molarratio of free cholesterol to phospholipids; immuno-affinity; content,activity or helicity of one or more of the following enzymes orproteins: ApoA-I, ApoA-II, ApoD, ApoE, ApoJ, ApoA-IV, cholesterol estertransfer protein (CETP), lecithin; cholesterol acyltransferase (LCAT);capacity and/or rate for cholesterol binding, capacity and/or rate forcholesterol transport.

The terms “modified high density lipoprotein” and “delipidated highdensity lipoprotein” may be used interchangeably and refer to reducedlipid blood products, and in particular, high density lipoproteinshaving a reduced lipid content, that may be contained within theresultant plasma once a delipidation process has been performed.Similarly, the term “treated plasma” refers to the resultant plasma oncea delipidation process has been performed.

FIG. 1A is a flow chart illustrating an exemplary process for separatingpre-beta HDL from modified HDL, in accordance with some embodiments ofthe present specification. At 102, a plasma delipidation process isstarted for a subject or a patient who is suffering from acardiovascular or a related disease. The process is typically startedafter one or more observations made by a physician treating the patient.The observations may be based on a combination of symptoms andtest-results such as but not limited to from blood tests andimaging-analyses. The observation may lead the physician to concludethat treatment for the patient requires reduction of harmful lipids fromthe patient's physiological system.

At 104, a blood fraction is obtained, which in an embodiment, is plasma.In accordance with embodiments of the present specification, the bloodfraction is obtained from either the patient (autologous) or from anon-autologous source. The blood fraction from the non-autologous sourceis collected from healthy, voluntary donors. The process of bloodfractionation is typically done by filtration, centrifuging the blood,aspiration, or any other method known to persons skilled in the art.Blood fractionation separates the plasma from the blood. In anembodiment, blood fractionation is performed remotely from the methoddescribed in context of FIG. 1A. In one embodiment, blood is withdrawnfrom a patient or a donor in a volume sufficient to produce about 12ml/kg of plasma based on body weight. During the fractionation process,the blood can optionally be combined with an anticoagulant, such assodium citrate, and centrifuged at forces approximately equal to 2,000times gravity. The blood is separated into plasma and red blood cellsusing methods commonly known to one of skill in the art, such asplasmapheresis. In an embodiment, the red blood cells are then aspiratedfrom the plasma. In one embodiment, the process of blood fractionationis performed by withdrawing blood from the patient with thecardiovascular and/or related disease, and who is being treated by thephysician. In an alternative embodiment, the process of bloodfractionation is performed by withdrawing blood from a person other thanthe patient with the cardiovascular and/or related disease who istreated by the physician. Therefore, the plasma obtained as a result ofthe blood fractionation process may be either autologous ornon-autologous.

Subsequent to fractionation, the red blood cells are either stored in anappropriate storage solution or, preferably, returned to the patientduring plasmapheresis. Physiological saline may also optionally beadministered to the patient to replenish volume. If the blood wasobtained from an individual other than the patient, the cells arereturned to that individual, who can also be referred to as the donor.

Plasma obtained from blood is usually a straw-colored liquid thatcomprises the extracellular matrix of blood cells. Plasma is typically95% water, and contains dissolved proteins, which constitute about 6-8%of plasma. The plasma also contains glucose, clotting factors,electrolytes, hormones, carbon dioxide, and oxygen. The plasma has adensity of approximately 1006 kg/m3, or 1.006 g/ml.

In some alternate embodiments, Low Density Lipoprotein (LDL) is alsoseparated from the plasma. Separated LDL is usually discarded. Inalternative embodiments, LDL is retained in the plasma. In accordancewith embodiments of the present specification, blood fraction or plasmaobtained at 104 includes plasma with High Density Lipoprotein (HDL), andmay or may not include other protein particles. In embodiments,autologous or non-autologous plasma collected from the patient or donor,respectively, is subsequently isolated via an approved plasmapheresisdevice. The plasma may be transported using a continuous or batchprocess.

At 106, the blood fraction or plasma obtained at 104 is mixed with oneor more solvents, such as lipid removing agents. In an embodiment, thesolvents used include either or both of organic solvents sevoflurane andn-butanol. In embodiments, the plasma and solvent are introduced into atleast one apparatus for mixing, agitating, or otherwise contacting theplasma with the solvent. In embodiments, the solvent system is optimallydesigned such that only the HDL particles are treated to reduce theirlipid levels and LDL levels are not affected. The solvent systemincludes factoring in variables such as the solvent employed, mixingmethod, time, and temperature. Solvent type, ratios and concentrationsmay vary in this step. Acceptable ratios of solvent to plasma includeany combination of solvent and plasma. In some embodiments, ratios usedare 2 parts plasma to 1 part solvent, 1 part plasma to 1 part solvent,or 1 part plasma to 2 parts solvent. In an embodiment, when using asolvent comprising 95 parts sevoflurane to 5 parts n-butanol, a ratio oftwo parts solvent per one part plasma is used. Additionally, in anembodiment employing a solvent containing n-butanol, the presentspecification uses a ratio of solvent to plasma that yields at least 3%n-butanol in the final solvent/plasma mixture. In an embodiment, a finalconcentration of n-butanol in the final solvent/plasma mixture is 3.33%.The plasma and solvent are introduced into at least one apparatus formixing, agitating, or otherwise contacting the plasma with the solvent.The plasma may be transported using a continuous or batch process.Further, various sensing means may be included to monitor pressures,temperatures, flow rates, solvent levels, and the like. The solventsdissolve lipids from the plasma. In embodiments of the presentspecification, the solvents dissolve lipids to yield treated plasma thatcontains modified HDL particles with reduced lipid content. The processis designed such that HDL particles are treated to reduce their lipidlevels and yield modified HDL particles without destruction of plasmaproteins or substantially affecting LDL particles. It should be notedthat there is no clinically significant decrease in blood constituentspost-plasmapheresis.

Energy is introduced into the system in the form of varied mixingmethods, time, and speed. At 108, bulk solvents are removed from themodified HDL particles via centrifugation. In embodiments, any remainingsoluble solvent is removed via charcoal adsorption, evaporation, orHollow Fiber Contractors (HFC) pervaporation. The mixture is optionallytested for residual solvent via use of Gas Chromatography (GC), orsimilar means. The test for residual solvent may optionally beeliminated based on statistical validation.

The extracted modified HDL solution has an increased concentration ofpre-beta HDL. It is estimated that the modified HDL in the delipidatedplasma, has approximately 80-85% of pre-β particles, and about 15-20% ofa HDL particles. Concentration of pre-beta HDL is greater in themodified HDL, relative to the original HDL that was present in theplasma before treating it with the solvent. Compared to the plasmasolution originally separated from the blood fraction, which typicallycontains approximately 5% of pre-β HDL particles, the concentration ofpre-β HDL particles is substantially increased.

FIG. 1B illustrates an exemplary embodiment of a system and itscomponents used to achieve the methods of the present specification. Thefigure depicts an exemplary basic component flow diagram definingelements of the HDL modification system 200. Embodiments of thecomponents of system 200 are utilized after obtaining a blood fractionfrom a patient or another individual (donor). The plasma, separated fromthe blood is brought in a sterile bag to system 200 for furtherprocessing. A fluid input 205 is provided and connected via tubing to amixing device 220. A solvent input 210 is provided and also connectedvia tubing to mixing device 220. In embodiments, valves 215, 216 areused to control the flow of fluid from fluid input 205 and solvent fromsolvent input 210 respectively. It should be appreciated that the fluidinput 205 contains any fluid that includes HDL particles, includingplasma having LDL particles or devoid of LDL particles, as discussedabove. It should further be appreciated that solvent input 210 caninclude a single solvent, a mixture of solvents, or a plurality ofdifferent solvents that are mixed at the point of solvent input 210.While depicted as a single solvent container, solvent input 210 cancomprise a plurality of separate solvent containers. Embodiments oftypes of solvents that may be used are discussed above.

Mixer 220 mixes fluid from fluid input 205 and solvent from solventinput 210 to yield a fluid-solvent mixture. In embodiments, mixer 220 iscapable of using a shaker bag mixing method with the input fluid andinput solvent in a plurality of batches, such as 1, 2, 3 or morebatches. An exemplary mixer is a Barnstead Labline orbital shaker table.Once formed, the fluid-solvent mixture is directed, through tubing andcontrolled by at least one valve 215 a, to a separator 225. In anembodiment, separator 225 is capable of performing bulk solventseparation through gravity separation in a funnel-shaped bag.

In separator 225, the fluid-solvent mixture separates into a first layerand second layer. The first layer comprises a mixture of solvent andlipid that has been removed from the HDL particles. The first layer istransported through a valve 215 b to a first waste container 235. Thesecond layer comprises a mixture of residual solvent, modified HDLparticles, and other elements of the input fluid. One of ordinary skillin the art would appreciate that the composition of the first layer andthe second layer would differ based upon the nature of the input fluid.Once the first and second layers separate in separator 225, the secondlayer is transported through tubing to a solvent extraction device 240.In an embodiment, a pressure sensor 229 and valve 230 is positioned inthe flow stream to control the flow of the second layer to solventextraction device 240.

The opening and closing of valves 215, 216 to enable the flow of fluidfrom input containers 205, 210 may be timed using mass balancecalculations derived from weight determinations of the fluid inputs 205,210 and separator 225. For example, the valve 215 b between separator225 and first waste container 235 and valve 230 between separator 225and solvent extraction device 240 open after the input masses (fluid andsolvent) substantially balances with the mass in separator 225 and asufficient period of time has elapsed to permit separation between thefirst and second layers. Depending on what solvent is used, andtherefore which layer settles to the bottom of separator 225, eithervalve 215 b between separator 225 and first waste container 235 isopened or valve 230 between separator 225 and solvent extraction device240 is opened. One of ordinary skill in the art would appreciate thatthe timing of the opening is dependent upon how much fluid is in thefirst and second layers and would further appreciate that it ispreferred to keep valve 215 b between separator 225 and first wastecontainer 235 open just long enough to remove all of the first layer andsome of the second layer, thereby ensuring that as much solvent aspossible has been removed from the fluid being sent to solventextraction device 240.

In embodiments, a glucose input 255 and one or more saline inputs 260are in fluid communication with the fluid path 221 leading fromseparator 225 to solvent extraction device 240. A plurality of valves215 c and 215 d are also incorporated in the flow stream from glucoseinput 255 and saline input 260 respectively, to the tubing providing theflow path 221 from separator 225 to solvent extraction device 240.Glucose and saline are incorporated into embodiments of the presentspecification in order to prime solvent extraction device 240 prior tooperation of the system. Where such priming is not required, the glucoseand saline inputs are not required. Also, one of ordinary skill in theart would appreciate that the glucose and saline inputs can be replacedwith other primers if solvent extraction device 240 requires it.

In some embodiments, solvent extraction device 240 is a charcoal columndesigned to remove the specific solvent used in solvent input 210. Anexemplary solvent extraction device 240 is an Asahi Hemosorber charcoalcolumn. A pump 250 is used to move the second layer from separator 225,through solvent extraction device 240, and to an output container 245.In embodiments, pump 250 is a rotary peristaltic pump, such as aMasterflex Model 77201-62.

The first layer is directed to waste container 235 that is in fluidcommunication with separator 225 through tubing and at least one valve215 b. Additionally, other waste, if generated, can be directed from thefluid path connecting solvent extraction device 240 and output container245 to a second waste container 255. Optionally, in an embodiment, avalve 215 f is included in the path from the solvent extraction device240 to the output container 245. Optionally, in an embodiment, a valve215 g is included in the path from the solvent extraction device 240 tothe second waste container 255.

In an embodiment of the present specification, gravity is used, whereverpractical, to move fluid through each of the plurality of components.For example, gravity is used to drain input plasma 205 and input solvent210 into mixer 220. Where mixer 220 comprises a shaker bag and separator225 comprises a funnel bag, fluid is moved from the shaker bag to thefunnel bag and, subsequently, to first waste container 235, ifappropriate, using gravity.

In an additional embodiment, not shown in FIG. 1B, the output fluid inoutput container 245 is subjected to a solvent detection system, orlipid removing agent detection system, to determine if any solvent, orother undesirable component, is in the output fluid. In one embodiment,the output fluid is subjected to sensors that are capable of determiningthe concentrations of solvents introduced in the solvent input, such asn-butanol or di-isopropyl ether. In embodiments, the sensors are capableof providing such concentration information on a real-time basis andwithout having to physically transport a sample of the output fluid, orair in the headspace, to a remote device.

In an embodiment, the output fluid is further processed, in a secondstage, to separate or to isolate at least pre-β HDL particles, and ifrequired then both α and pre-β HDL particles. In an embodiment, thesecond stage (as described below) occurs in a separate and discrete areafrom the delipidation process where the end product output fluid istransported to a processing lab or room. In an alternate embodiment, thesecond stage processing occurs in-line with the delipidation system,whereby the system is connected to an affinity column sub-system orultracentrifugation sub-system. The resultant separated a and/or pre-βHDL particles are then introduced to the bloodstream of the patient.

In one embodiment, molecularly imprinted polymer technology is used toenable surface acoustic wave sensors. A surface acoustic wave sensorreceives an input, through some interaction of its surface with thesurrounding environment, and yields an electrical response, generated bythe piezoelectric properties of the sensor substrate. To enable theinteraction, molecularly imprinted polymer technology is used.Molecularly imprinted polymers are plastics programmed to recognizetarget molecules, like pharmaceuticals, toxins or environmentalpollutants, in complex biological samples. The molecular imprintingtechnology is enabled by the polymerization of one or more functionalmonomers with an excess of a crosslinking monomer in presence of atarget template molecule exhibiting a structure similar to the targetmolecule that is to be recognized, i.e. the target solvent.

The use of molecularly imprinted polymer technology to enable surfaceacoustic wave sensors can be made more specific to the concentrations oftargeted solvents and are capable of differentiating such targetedsolvents from other possible interferents. As a result, the presence ofacceptable interferents that may have similar structures and/orproperties to the targeted solvents would not prevent the sensor fromaccurately reporting existing respective solvent concentrations.

Alternatively, if the input solvent comprises certain solvents, such asn-butanol, electrochemical oxidation could be used to measure thesolvent concentration. Electrochemical measurements have severaladvantages. They are simple, sensitive, fast, and have a wide dynamicrange. The instrumentation is simple and not affected by humidity. Inone embodiment, the target solvent, such as n-butanol, is oxidized on aplatinum electrode using cyclic voltammetry. This technique is based onvarying the applied potential at a working electrode in both the forwardand reverse directions, at a predefined scan rate, while monitoring thecurrent. One full cycle, a partial cycle, or a series of cycles can beperformed. While platinum is the preferred electrode material, otherelectrodes, such as gold, silver, iridium, or graphite, could be used.Although, cyclic voltammetric techniques are used, other pulsetechniques such as differential pulse voltammetry or square wavevoltammetry may increase the speed and sensitivity of measurements.

Embodiments of the present specification expressly cover any and allforms of automatically sampling and measuring, detecting, and analyzingan output fluid, or the headspace above the output fluid. For example,such automated detection can be achieved by integrating a mini-gaschromatography (GC) measuring device that automatically samples air inthe output container, transmits it to a GC device optimized for thespecific solvents used in the delipidation process, and, using known GCtechniques, analyzes the sample for the presence of the solvents.

Referring back to FIG. 1B, suitable materials for use in any of theapparatus components as described herein include materials that arebiocompatible, approved for medical applications that involve contactwith internal body fluids, and in compliance with U.S. PVI or ISO 10993standards. Further, the materials do not substantially degrade from, forinstance, exposure to the solvents used in the present invention, duringat least a single use. The materials are sterilizable either byradiation or ethylene oxide (EtO) sterilization. Such suitable materialsare capable of being formed into objects using conventional processes,such as, but not limited to, extrusion, injection molding and others.Materials meeting these requirements include, but are not limited to,nylon, polypropylene, polycarbonate, acrylic, polysulfone,polyvinylidene fluoride (PVDF), fluoroelastomers such as VITON,available from DuPont Dow Elastomers L.L.C., thermoplastic elastomerssuch as SANTOPRENE, available from Monsanto, polyurethane, polyvinylchloride (PVC), polytetrafluoroethylene (PTFE), polyphenylene ether(PFE), perfluoroalkoxy copolymer (PFA), which is available as TEFLON PFAfrom E.I. du Pont de Nemours and Company, and combinations thereof.

Valves 215, 215 a, 215 b, 215 c, 215 d, 215 e, 215 f, 215 g, 216 and anyother valve used in each embodiment may be composed of, but are notlimited to, pinch, globe, ball, gate or other conventional valves. Insome embodiments, the valves are occlusion valves such as AcroAssociates' Model 955 valve. However, the present specification is notlimited to a valve having a particular style. Further, the components ofeach system described in accordance with embodiments of the presentspecification may be physically coupled together or coupled togetherusing conduits that may be composed of flexible or rigid pipe, tubing orother such devices known to those of ordinary skill in the art.

FIG. 1C illustrates an exemplary configuration of a system used inaccordance with some embodiments of the present specification to achievethe processes disclosed herein. Referring to FIG. 1C, a configuration ofbasic components of the HDL modification system 300 is shown. A fluidinput 305 is provided and connected via tubing to a mixing device 320. Asolvent input 310 is provided and also connected via tubing to a mixingdevice 320. Preferably valves 316 are used to control the flow of fluidfrom fluid input 305 and solvent from solvent input 310. It should beappreciated that the fluid input 305 preferably contains any fluid thatincludes HDL particles, including plasma having LDL particles or devoidof LDL particles, as discussed above. It should further be appreciatedthat solvent input 310 can include a single solvent, a mixture ofsolvents, or a plurality of different solvents that are mixed at thepoint of solvent input 310. While depicted as a single solventcontainer, solvent input 310 can comprise a plurality of separatesolvent containers. The types of solvents that are used and preferredare discussed above.

The mixer 320 mixes fluid from fluid input 305 and solvent from solventinput 310 to yield a fluid-solvent mixture. Preferably, mixer 320 iscapable of using a shaker bag mixing method with the input fluid andinput solvent in a plurality of batches, such as 1, 2, 3 or morebatches. Once formed, the fluid-solvent mixture is directed, throughtubing and controlled by at least one valve 321, to a separator 325. Ina preferred embodiment, separator 325 is capable of performing bulksolvent separation through gravity separation in a funnel-shaped bag.

In the separator 325, the fluid-solvent mixture separates into a firstlayer and second layer. The first layer comprises a mixture of solventand lipid that has been removed from the HDL particles. The second layercomprises a mixture of residual solvent, modified HDL particles, andother elements of the input fluid. One of ordinary skill in the artwould appreciate that the composition of the first layer and the secondlayer would differ based upon the nature of the input fluid. Once thefirst and second layers separate in separator 325, the second layer istransported through tubing to a solvent extraction device 340.Preferably, a pressure sensor 326 and valve 327 is positioned in theflow stream to control the flow of the second layer to the solventextraction device 340.

Preferably, a glucose input 330 and saline input 350 is in fluidcommunication with the fluid path leading from the separator 325 to thesolvent extraction device 340. A plurality of valves 331 is alsopreferably incorporated in the flow stream from the glucose input 330and saline input 350 to the tubing providing the flow path from theseparator 325 to the solvent extraction device 340. Glucose and salineare incorporated into the present invention in order to prime thesolvent extraction device 340 prior to operation of the system. Wheresuch priming is not required, the glucose and saline inputs are notrequired. Also, one of ordinary skill in the art would appreciate thatthe glucose and saline inputs can be replaced with other primers if thesolvent extraction device 340 requires it.

The solvent extraction device 340 is preferably a charcoal columndesigned to remove the specific solvent used in the solvent input 310.An exemplary solvent extraction device 340 is an Asahi Hemosorbercharcoal column. A pump 335 is used to move the second layer from theseparator 325, through the solvent extraction device 340, and to anoutput container 315. The pump is preferably a peristaltic pump, such asa Masterflex Model 77201-62.

The first layer is directed to a waste container 355 that is in fluidcommunication with separator 325 through tubing and at least one valve356. Additionally, other waste, if generated, can be directed from thefluid path connecting solvent extraction device 340 and output container315 to waste container 355.

Preferably, an embodiment of the present invention uses gravity,wherever practical, to move fluid through each of the plurality ofcomponents. For example, preferably gravity is used to drain the inputplasma 305 and input solvent 310 into the mixer 320. Where the mixer 320comprises a shaker bag and separator 325 comprises a funnel bag, fluidis moved from the shaker bag to the funnel bag and, subsequently, to thewaste container 355, if appropriate, using gravity.

In general, the present invention preferably comprises configurationswherein all inputs, such as input plasma and input solvents, disposableelements, such as mixing bags, separator bags, waste bags, solventextraction devices, and solvent detection devices, and output containersare in easily accessible positions and can be readily removed andreplaced by a technician.

To enable the operation of the above described embodiments of thepresent invention, it is preferable to supply a user of such embodimentswith a packaged set of components, in kit form, comprising eachcomponent required to practice embodiments of the present specification.The kit may include an input fluid container (i.e. a high densitylipoprotein source container), a lipid removing agent source container(i.e. a solvent container), disposable components of a mixer, such as abag or other container, disposable components of a separator, such as abag or other container, disposable components of a solvent extractiondevice (i.e. a charcoal column), an output container, disposablecomponents of a waste container, such as a bag or other container,solvent detection devices, and, a plurality of tubing and a plurality ofvalves for controlling the flow of input fluid (high densitylipoprotein) from the input container and lipid removing agent (solvent)from the solvent container to the mixer, for controlling the flow of themixture of lipid removing agent, lipid, and particle derivative to theseparator, for controlling the flow of lipid and lipid removing agent toa waste container, for controlling the flow of residual lipid removingagent, residual lipid, and particle derivative to the extraction device,and for controlling the flow of particle derivative to the outputcontainer.

In one embodiment, a kit comprises a plastic container having disposablecomponents of a mixer, such as a bag or other container, disposablecomponents of a separator, such as a bag or other container, disposablecomponents of a waste container, such as a bag or other container, and,a plurality of tubing and a plurality of valves for controlling the flowof input fluid (high density lipoprotein) from the input container andlipid removing agent (solvent) from the solvent container to the mixer,for controlling the flow of the mixture of lipid removing agent, lipid,and particle derivative to the separator, for controlling the flow oflipid and lipid removing agent to a waste container, for controlling theflow of residual lipid removing agent, residual lipid, and particlederivative to the extraction device, and for controlling the flow ofparticle derivative to the output container. Disposable components of asolvent extraction device (i.e. a charcoal column), the input fluid, theinput solvent, and solvent extraction devices may be providedseparately.

Extracting modified HDL by delipidating plasma may be referred to as afirst stage of the methods described in embodiments of the presentspecification. In accordance with embodiments of the presentspecification, at 110, the delipidated plasma is preserved forsubsequent, later use. In an embodiment, the delipidated plasma may beused after a prolonged time period, such as at least one year and morepreferably at least two years. In an embodiment, the delipidated plasmamay be stored for a predetermined time period, wherein the time periodmay be dependent on the preservation process employed.

The delipidated plasma (DP), in any aliquot or volume, may be preservedby utilizing suitable preservation methods, such as but not limited tofreezing. In various embodiments, any means of preservation may beemployed as long as the method retains a predefined amount of efficacyof the final product when compared to the freshly delipidated plasma.Accordingly, it is essential that the delipidated plasma, comprisingpre-beta HDL, be evaluated using 2D gel spot testing or quality testingto certify that the pre-beta HDL has not degraded to free Apo A1. Morespecifically, the present invention subjects a predefined portion of thestored delipidated plasma, comprising pre-beta HDL, to 2D gel testingand certifies that the batch associated with the tested delipidatedplasma is acceptable for administration to a patient if no more than 80%of the pre-beta HDL has degraded to Apo A1.

Referring to FIG. 2, a process 400 for acquiring, preserving, andthawing extracted delipidated plasma is shown. Extracted delipidatedplasma, comprising pre-beta HDL, is obtained 405 using the processesdescribed herein. Portions of a given batch of extracted delipidatedplasma are spot tested 410 to establish baseline amounts orconcentrations of pre-beta HDL. The spot testing 410 may be done using2D gel electrophoresis in which a sample of the batch of extracteddelipidated plasma is solubilized, loaded onto a gel, and subjected toan electric field which causes a movement of proteins, in accordancewith their isoelectric points, through the gel. The separated proteinsare then solubilized again and separated by their molecular weights onan orthogonal second axis. The spot testing 410 therefore quantifies aconcentration or amount of proteins, such as pre-beta HDL, in the samplealong two axes: isoelectric point and molecular weight. Other clinicalevaluations are further described below.

Once spot tested 410, batches are subjected to preservation 420,preferably through flash freezing as further described below. In oneembodiment, prior to preservation 420, the batch is optionally modified415 to insure that the protein concentration of pre-beta HDL is within apredefined range, as further described below. It has been determinedthat preserved pre-beta HDL is less stable if the concentration of thepreparation is too dilute and has decreased efficacy if theconcentration of the preparation is too high. Accordingly, the batch isoptionally modified, by dilution or concentration, 415 to insure thatthe concentration of pre-beta HDL in the delipidated plasma is within arange of 0.5 mg/dl to 500 mg/dl and preferably in a range of 1 mg/dl to400 mg/dl, or any increment therein. Preferably, the concentration ofpre-beta HDL in the delipidated plasma is no greater than 500 mg/dl.

After preservation 420, the delipidated plasma is stored, which can befor a week up to 3 years or any increment therein. At some point, thedelipidated plasma is thawed 430. In one embodiment, thawing is achievedby taking the frozen delipidated plasma and placing it an environmenthaving a temperature range of 2° C. to 26° C. In one embodiment, thethawing delipidated plasma is kept in an environment having atemperature range of 3° C. to 5° C. and more preferably 4° C. for aperiod of no more than 48 hours. Preferably, the thawed delipidatedplasma is used within a 48 hour period of thawing, and more preferablywithin a 24 hour period, and is not re-frozen again. After thawing 430,a portion of the batch may be tested again 440 to determine if thepre-beta HDL in the delipidated plasma has degraded or is no longereffective, as further described below.

Optionally an additive may be included as part of the preservationprocess. An additive may be added to either the precursor or finalproduct in order to enhance the preservation process. In embodiments,the DP is preserved using methods and standards similar to thoseapplicable for preserving plasma. In an embodiment, the preservation isachieved by freezing. Some of these standard methods and practices aredefined in the CFR and the AABB, ABC, ARC circular of Information forthe Use of Human Blood and Blood Components, and by EuropeanPharmacopeia guidelines for preparation of plasma for manufacturing. Avolume or aliquot of the delipidated source plasma is placed in afreezer within a few hours of completing the delipidation process. Insome embodiments, the DP is placed in a freezer within 8 hours of thedelipidation process, or within a timeframe specified in the directionsfor use for the blood collecting, processing, and storage system. Inembodiments, the DP volume is frozen per standard means for fresh frozenplasma at a temperature of approximately −18° C. to −80° C.

It should be noted herein that the volumes or aliquots indicated aboveare only exemplary and that any amount (volume or aliquot) of adelipidated plasma sample may be preserved using the systems and methodsof the present specification. It should also be noted that multiplevolumes or aliquots may be preserved either in series, where each volumeor aliquot is sequentially preserved, or in parallel where multiplevolumes or aliquots are preserved simultaneously.

Referring back to the preservation step, in one embodiment, and by wayof example, 50 ml of delipidated plasma (DP) is frozen via flashfreezing method using liquid nitrogen followed by storage at −80° C. Inanother embodiment, and by way of example, 50 ml of delipidated plasma(DP) is frozen using a slower freezing method at −80° C. In yet anotherembodiment, and by way of example, 50 ml of delipidated plasma (DP) isfrozen using a slower freezing method at −20° C. in a frost-freefreezer. In one embodiment, and by way of example, 100 ml of DP isfrozen using a flash freezing method. In another embodiment, and by wayof example, up to 400 ml of DP is frozen using a flash freezing methods.In other embodiments, DP volumes (units) ranging from at least 1 ml to400 ml or higher volumes are frozen together using the preservationmethods described above; any method of freezing may be used to preserveany volume of DP. The time needed to freeze a sample is size dependent;therefore, the amount of freezing time is different for differentaliquot sizes. In embodiments, the time taken to freeze an aliquot sizeis also a function of the method used for freezing. In embodiments, thetime needed to freeze a sample is predetermined and based on sample sizeand/or freezing method. In one embodiment, the time needed to flashfreeze concentrated pre-beta HDL to −80° C. is less than 30 minutes,less than 20 minutes, and preferably less than 10 minutes. In anotherembodiment, the temperature for flash freezing is less than −30° C.

Other methods of freezing may be used in various embodiments of thepresent specification. The selected method of freezing would ensure thatcritical components of the DP are maintained. The duration of expirationof frozen delipidated plasma may vary for different freezingtemperatures. Generally, for colder freezing temperatures, the productmay be stored for a longer duration and thus, has a longer “shelf life”(slower expiration).

In embodiments, each unit of source plasma is assessed individuallybefore and after the delipidation process. The plasma is assessed forparameters, including but not limited to the following parameters:

-   -   1. Concentration and size of pre-β and a HDL particles. In some        embodiments, 2D gel electrophoresis technique is used with both        heavy and light gels and immunoblotting for ApoA-I.    -   2. Clinical chemistry of the plasma. Various characteristics        determined may include total cholesterol, HDL, LDL, ApoA-I,        ApoB, triglycerides, CBC, sodium, potassium, chloride, calcium,        phosphorous, creatinine, BUN, fibrinogen, aPTT, PT, ALT, AST,        ALP, bilirubin, uric acid, glucose, LDH    -   3. Additionally, fractions of the DP, before freezing and after        freezing and thawing in a subsequent step, is assessed for        cholesterol content by UV absorbance using known techniques such        as Fast Protein Liquid Chromatography (FPLC)).    -   4. Selective samples/fractions will be assayed for cholesterol        efflux capacity, which include, but are not limited to the        fractions delineated in FIGS. 1A and 1G.

In embodiments, the parameters described above are also assessed beforepreserving or freezing of the delipidated plasma and after thawing ofthe delipidated plasma. In some embodiments, the efficacy of the thaweddelipidated plasma is in the range of 1% to 100% of the efficacy of thepre-preservation delipidated plasma. In some embodiments, the efficacyof the thawed delipidated plasma is in the range of 1% to 150% of theefficacy of the pre-preservation delipidated plasma. In someembodiments, the efficacy of the thawed delipidated plasma is in therange of 1% to 200% of the efficacy of the pre-preservation delipidatedplasma. In some embodiments, the efficacy of the thawed delipidatedplasma is lower than the efficacy of the pre-preservation delipidatedplasma. In some embodiments, the efficacy of the thawed delipidatedplasma is greater than the efficacy of the pre-preservation delipidatedplasma.

At 112, the preserved DP, is prepared for normal use for furtherprocessing or for treating a patient. The patient treated by the DP mayor may not be the individual from who the plasma was obtained for thedelipidation process (it may be autologous or non-autologous). If, at110, the DP was preserved by freezing, then at 112 it is prepared bythawing. In an embodiment, the frozen DP is thawed with a water bath ata temperature in the range of 30° C. to 37° C. for approximately 30minutes. In some embodiments, the thawed plasma is maintained at 1° C.to 6° C. for 1-5 days. In one more embodiment, the frozen DP is thawedslowly at room temperature. In another embodiment, the frozen DP isthawed rapidly in a refrigerator at approximately 5° C. Other methods ofthawing frozen DP may be utilized for different quantities andcompositions of DP.

In various embodiments of the present specification, in a second stage,delipidated or modified HDL is further treated to separate or isolatecomponents of HDL particles such as pre-β HDL particles or a combinationof α and pre-β HDL particles. At 114, the treated and thawed plasmacontaining modified HDL particles with reduced lipid content, which wasseparated from the solvents at 108, is further treated with solvents toyield a solution comprising a higher concentration of α and pre-β HDLparticles. Exemplary methods for separating alpha and pre-beta particlesfrom delipidated plasma are discussed below.

At 116, in accordance with some optional embodiments of the presentspecification, the separated α and pre-β HDL particles are preserved byfreezing and may be used after a prolonged period after thawing at 118.In one embodiment, thawing is achieved by taking the frozen delipidatedplasma and placing it an environment having a temperature range of 2° C.to 26° C. In one embodiment, the thawing delipidated plasma is kept inan environment having a temperature range of 3° C. to 5° C. and morepreferably 4° C. for a period of no more than 48 hours. Preferably, thethawed delipidated plasma is used within a 48 hour period of thawing,and more preferably within a 24 hour period, and is not re-frozen again.In embodiments, the process of preserving and preparing derived α andpre-β HDL particles is similar to the methods for preserving andpreparing DP. In an embodiment, flash freezing methods are preferablyused to freeze derived α and pre-β HDL particles.

Methods of Separating Pre-Beta Particles from Delipidated Plasma

In an embodiment, affinity chromatography may be used to reduce theamount of unwanted substances in delipidated plasma (for example, plasmaproteins and certain lipoproteins, such as LDL and VLDL), and thereforeincrease the concentration of a desired substance (for example, pre-βHDL particles).

FIG. 1D is a flow chart illustrating an exemplary set of steps that areused to increase the concentration of desired substances in the treatedplasma, using affinity chromatography, in accordance with someembodiments of the present specification. In an embodiment, an affinitycolumn is used to entrap ApoA-I protein particles (and therefore pre-βHDL) particles while removing undesired particles from the treatedplasma. At 120, treated plasma is added to the column for affinitychromatography utilizing an antibody to ApoA-I so that ApoA-I is boundto the antibody when the treated plasma is run through the column. Asolid medium, such as a resin, may be used to bind the desired substancein the form of HDL particles. The desired HDL particles may comprise amixture of α-HDL and pre-β HDL particles. The composition of theseparticles is on the order of approximately 15% α-HDL and 85% pre-βparticles. At 122, the unbound and unwanted substances, including LDL,and VLDL drip through the column (as they do not bind to the antibody toApoA-I), and are thereby removed from the treated plasma solution. At124, a washing buffer may be run through the column to remove anyunwanted proteins. A disassociating reagent or solution is then runthrough the column so that the pre-β HDL particles (or ApoA-I) whichcontains the pre-β HDL particles) are no longer bound to the antibody toApoA-I and are effectively separated.

Ultracentrifugation is another method that may be employed to reduce theamount of unwanted substances (for example, plasma proteins and certainlipoproteins, such as LDL and VLDL, and in this case, a HDL particles),and therefore increase the concentration of desired substance (forexample, pre-β HDL particles). In this method, the principle ofcentrifugation is utilized to separate constituents of a solution byrotating the solution at very high speeds.

The starting material delipidated plasma solution has a density of 1006kg/m3, or 1.006 g/ml. In order to separate out the various fractionsduring ultracentrifugation, as discussed below, it may be necessary toadjust the density of the starting material at each step. Inembodiments, the density can be adjusted by adding a volume of a moredense substance or solvent (such as Potassium Bromide (KBr) to thetreated plasma. In an embodiment, any solution having a density that canbe used to adjust the density of the starting material may be employed.

In an embodiment, a concentrated stock solution of Potassium Bromide(KBr) and Sodium Chloride (NaCl), having a combined density of 1.346 isadded to the delipidated/treated plasma solution to adjust the densityof the treated plasma solution to a desired density. The stock solutionhas a density higher than 1.25 g/mL (for example, an aqueous solution of4.62M KBr, has a density of 1.37 g/mL), while the treated plasmasolution has a density of approximately 1.006 g/mL. The solutionresulting from the addition of a dense substance to treated plasma willhave a specific density that will allow for separation of the variousfractions during ultracentrifugation. Methods for adjusting density arewell known to those of ordinary skill in the art and also described inHavel et al., J. Clin. Invest. 1955, 34; 1345-1353, which discusses amethod for separating and purifying lipoprotein fractions on apreparative scale for analysis and use in metabolic studies and which isherein incorporated by reference.

In an embodiment, the combined plasma and solvent are introduced into anultracentrifugation tube (or may be combined in the tube itself). Thetubes may be held by a rotor. The tubes may be spun at a high speed forsufficient time to produce a separation, following which the rotor comesto a smooth stop and a gradient is gently pumped out of each tube toisolate the separated components. In one embodiment, ultracentrifugationis performed at speeds of approximately 105,000×g and for roughly 16-20hours. It should be noted that the spin time is adjusted based on thedensity of the starting material and the relative densities of thedesired fractions that are to be separated out For example, if densityof the material that is extracted in a first step is 1.21 g/mL and asubsequent density that is desired in a second step is 1.25 g/mL, itwill take longer to separate out the two materials of similar densitythat it would take to separate out a density of 1.006 g/mL from astarting material having a density of 1.21 g/mL.

In embodiments, the resultant desired fraction includes at least pre-βHDL particles which are separated from the treated plasma.

FIG. 1E is a flow chart illustrating an exemplary set of steps that areused to increase the concentration of desired substances in the treatedplasma, using ultracentrifugation, in accordance with some embodimentsof the present specification. In an embodiment, the method described inthis flow chart may be used to more quickly separate pre-β HDL usingultracentrifugation. At 130, a treated plasma starting material can bespun at an adjusted density of 1.21, which corresponds to the density ofHDL. The top fraction will contain VLDL, LDL, and a HDL, while pre-β HDLwill be in the bottom fraction. In an embodiment, the spinning isperformed over a period of 18-24 hours. At 132, the bottom fractioncontaining pre-β HDL and plasma proteins is isolated. At 134, theisolated bottom fraction containing pre-β HDL is spun at a density of1.25 yielding a top fraction containing pre-β HDL and a bottom fractioncontaining plasma proteins. In an embodiment, the spinning is performedover a period of 24-48 hours. At 136, the resultant top bottom fractioncontaining pre-β HDL is isolated from the bottom fraction containingplasma proteins, yielding a concentrated pre-β HDL product.

At the end of the process of ultracentrifugation, pre-β HDL particlesfloat to the top, and may be separated to obtain a solution with a highconcentration of pre-β HDL particles. Subsequently, the tube containingconcentrated pre-β HDL particles may be detached.

FIG. 1F is a flow chart illustrating another exemplary set of steps thatare used to increase the concentration of desired substances in thetreated plasma, using ultracentrifugation, in accordance with someembodiments of the present specification.

In some embodiments, ultracentrifugation is a stepwise process that isperformed at different densities. Since lipoproteins are lighter thanproteins, it is possible to separate them from the delipidated (treated)plasma using ultracentrifugation at different densities to achievesequential fractionation. At 140, the delipidated (treated) plasma isspun at a density of 1.006 for 18-24 hours, which corresponds to thedensity of plasma (1006 kg/m3, or 1.006 g/ml). At this stage, theproteins are at the bottom of the centrifugation tube, while VLDLresides at the top. In addition, plasma containing LDL and HDL is at thebottommost portion of the tube.

At step 142, the fraction containing the plasma with LDL/HDL isseparated from the other constituents in the tube.

At step 144, the plasma containing LDL/HDL (plus a solution to adjustthe density) is spun at a density of 1.063, which corresponds to thedensity of LDL. The result is a fraction containing LDL at the top and afraction containing HDL at the bottom of the tube.

At step 146, the bottom fraction containing the HDL is separated fromthe other constituents in the tube.

At step 148, the HDL fraction is spun at a density of 1.21, whichcorresponds to the density of HDL. The end result is a fractioncontaining HDL at the top and pre-beta HDL at the bottom of the tube.

At step 150, the bottom fraction containing the pre-beta HDL is spun ata density of 1.25. The end result is a fraction containing pre-beta HDLat the top of the tube and the remaining plasma constituents at thebottom of the tube. At the end of the process of ultracentrifugation,pre-β HDL particles float to the top, and may be separated to obtain asolution with a high concentration of pre-β HDL particles. Subsequently,the tube containing concentrated pre-β HDL particles may be detached.The isolated lipoprotein fractions are separated from KBr usingdialysis, gel filtration or other suitable methodologies.

It may be understood by persons skilled in the art that the fractionsderived through spinning the treated plasma can be separated indifferent ways, and is not limited to the methods described in contextof FIGS. 1E and 1F.

FIG. 1G is a flow chart illustrating another exemplary set of steps thatare used to increase the concentration of desired substances in thetreated plasma, using ultracentrifugation, in accordance with someembodiments of the present specification. In an embodiment, the methoddescribed in this flow chart may be used to more quickly separate both α(alpha-1) and pre-β HDL from delipidated plasma usingultracentrifugation. At 170, a treated plasma starting material can bespun at an adjusted density of 1.063, which corresponds to the densityof LDL. The result is a fraction containing LDL at the top and afraction containing HDL at the bottom of the tube. In an embodiment, thespinning is performed over a period of 18-24 hours. At 172, the bottomfraction containing alpha-1 and pre-β HDL and plasma proteins isisolated. At 174, the isolated bottom fraction containing alpha-1 andpre-β HDL is spun at a density of 1.25 yielding a top fractioncontaining alpha-1 and pre-β HDL and a bottom fraction containing plasmaproteins. In an embodiment, the spinning is performed over a period of24-48 hours. At 176, the resultant top fraction containing alpha-1 andpre-β HDL is isolated from the bottom fraction containing plasmaproteins, yielding a concentrated product comprising alpha-1 and pre-βHDL.

At the end of the process of ultracentrifugation, alpha-1 and pre-β HDLparticles float to the top, and may be separated to obtain a solutionwith a high concentration of alpha-1 and pre-β HDL particles.Subsequently, the tube containing concentrated alpha-1 and pre-β HDLparticles may be detached. The isolated lipoprotein fractions areseparated from KBr by dialysis, gel filtration or other suitablemethodologies.

Referring back to FIG. 1A, at 160, the treated plasma containingconcentrated solution of pre-β HDL or a combination of α and pre-β HDLparticles with reduced lipid content is separated from the solvents,treated appropriately, and subsequently delivered to the patient. Thedelivered solution has a further increased concentration of α and/orpre-beta HDL. The resulting treated plasma containing the HDL particleswith reduced lipid and increased pre-beta concentration is provided tothe patient following dialysis with saline. In case of affinitychromatography, dialysis also releases entrapped desired substances(concentrated pre-β HDL particles). In case of autologous plasma, if thered cells of the patient were not already returned duringplasmapheresis, they may be administered to the patient at some pointduring the procedure. In an optional embodiment, the red blood cells maybe returned to the patient after combining them with the isolated αand/or pre-β HDL particles. One route of administration is through thevascular system, preferably intravenously.

In another alternative embodiment, a two stage process is applied tocompletely isolate pre-β HDL particles. First, affinity chromatographyis used to separate a solution comprising both pre-β and a HDLparticles. Following which, ultracentrifugation is used to fully isolatepre-β HDL particles.

The above examples are merely illustrative of the many applications ofthe system of present invention. Although only a few embodiments of thepresent invention have been described herein, it should be understoodthat the present invention might be embodied in many other specificforms without departing from the spirit or scope of the invention.Therefore, the present examples and embodiments are to be considered asillustrative and not restrictive, and the invention may be modifiedwithin the scope of the appended claims.

We claim:
 1. A method for preserving pre-beta high density lipoproteinfor administration to a patient, comprising: obtaining a batch ofdelipidated plasma comprising the pre-beta high density lipoprotein;testing a portion of the batch of the delipidated plasma to characterizethe pre-beta high density lipoprotein; preserving the batch of thedelipidated plasma; preparing the preserved delipidated plasma foradministration to the patient; testing the prepared delipidated plasmato characterize the pre-beta high density lipoprotein; and administeringthe pre-beta high density lipoprotein to the patient.
 2. The method ofclaim 1, further comprising, prior to preserving, modifying an amount ofthe pre-beta high density lipoprotein to insure a concentration of thepre-beta high density lipoprotein is in a range of 1 mg/dl to 400 mg/dl.3. The method of claim 1, wherein preserving comprises freezing thebatch at a temperature less than −30° C.
 4. The method of claim 1,wherein preparing comprises thawing the preserved delipidated plasma ina temperature range of 2° C. to 26° C.
 5. The method of claim 1, whereinpreserving comprises subjecting a volume of delipidated plasma in arange from 1 milliliter to 2 liters to a temperature less than −30° C.for less than 20 minutes.
 6. The method of claim 1, wherein testing theportion of the batch of the delipidated plasma to characterize thepre-beta high density lipoprotein comprises determining a firstconcentration of the pre-beta high density lipoprotein.
 7. The method ofclaim 6, wherein testing the prepared delipidated plasma to characterizethe pre-beta high density lipoprotein comprises determining a secondconcentration of the pre-beta high density lipoprotein and comparing thesecond concentration of the pre-beta high density lipoprotein to thefirst concentration of the pre-beta high density lipoprotein todetermine an extent of degradation.
 8. The method of claim 7, furthercomprising determining if the prepared delipidated plasma is suitablefor administration based on the second concentration of the pre-betahigh density lipoprotein.
 9. The method of claim 1, further comprising,prior to preservation, adding a preservative to the delipidated plasma.10. The method of claim 1, wherein preparing comprises thawing thepreserved delipidated plasma and further comprising storing the thaweddelipidated plasma at a temperature in a range of 1° C. to 6° C. for nomore than 5 days.
 11. A method for preserving modified high densitylipoproteins for administration to a patient, comprising: obtaining abatch of delipidated plasma comprising the modified high densitylipoproteins by connecting at least one person to a device forwithdrawing blood, withdrawing blood containing blood cells from the atleast one person, separating the blood cells from the blood to yield ablood plasma fraction containing high density lipoproteins and lowdensity lipoproteins, delipidating the high density lipoproteins using asolvent, separating out the low density lipoproteins, and collecting thedelipidated plasma with the modified high density lipoproteins; testinga portion of the batch of the delipidated plasma to characterize themodified high density lipoproteins; preserving the batch of thedelipidated plasma; preparing the preserved delipidated plasma foradministration to the patient; testing the prepared delipidated plasmato characterize the modified high density lipoproteins; andadministering the modified high density lipoproteins to the patient. 12.The method of claim 11, further comprising, prior to preserving,modifying an amount of the modified high density lipoproteins to insurea concentration of the modified high density lipoproteins is in a rangeof 1 mg/dl to 400 mg/dl.
 13. The method of claim 11, wherein preservingcomprises freezing the batch at a temperature less than −30° C.
 14. Themethod of claim 11, wherein preparing comprises thawing the preserveddelipidated plasma in a temperature range of 2° C. to 26° C.
 15. Themethod of claim 11, wherein preserving comprises subjecting a volume ofdelipidated plasma in a range from 1 milliliter to 2 liters to atemperature less than −30° C. for less than 20 minutes.
 16. The methodof claim 11, wherein testing the portion of the batch of the delipidatedplasma to characterize the modified high density lipoproteins comprisesdetermining a first concentration of the modified high densitylipoproteins.
 17. The method of claim 16, wherein testing the prepareddelipidated plasma to characterize the modified high densitylipoproteins comprises determining a second concentration of themodified density lipoproteins and comparing the second concentration ofthe modified high density lipoproteins to the first concentration of themodified density lipoproteins to determine an extent of degradation. 18.The method of claim 17, further comprising determining if the prepareddelipidated plasma is suitable for administration based on the secondconcentration of the modified high density lipoproteins.
 19. The methodof claim 11, further comprising, prior to preservation, adding apreservative to the delipidated plasma.
 20. The method of claim 11,wherein preparing comprises thawing the preserved delipidated plasma andfurther comprising storing the thawed delipidated plasma at atemperature in a range of 1° C. to 6° C. for no more than 5 days.